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G L Stiles

Publications and source records attributed to G L Stiles.

At least 73 records · Page 4Linked to original sources
PubMed1990

Purification and characterization of bovine cerebral cortex A1 adenosine receptor.

A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine greater than (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA greater than NECA greater than S-PIA. The A1AR was solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy]-phenyl]-1,3- dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand [( 3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax greater than 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA greater than S-PIA greater than NECA. The overall yield from soluble preparation was 7%. The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to alpha-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes.

Animals↗
PubMed1990

Agonists and antagonists recognize different but overlapping populations of A1 adenosine receptors: modulation of receptor number by MgCl2, solubilization, and guanine nucleotides.

A1 selective agonist and antagonist radioligands bind to the same A1 adenosine receptor binding subunit, as documented by photoaffinity labelling and partial peptide maps. In this study we document that although these radioligands recognize the same A1 adenosine receptor (A1AR), they recognize different numbers of A1ARs in bovine brain membranes, with agonist number being greater than antagonist number. Neither addition of guanine nucleotides nor removal of Mg2+ ions enhanced antagonist binding in membranes. On solubilization, agonists still recognized a greater number of A1ARs but addition of guanine nucleotides or removal of Mg2+ substantially increased the number of receptors detected with antagonist radioligands. The effects of Mg2+ and guanine nucleotides were not additive, suggesting that formation of a "low agonist-receptor-G protein state" by either modulating agent was sufficient to alter the receptor conformation such that it could be recognized by antagonist. These studies suggest that a proportion of the "precoupled A1AR-G protein complex" in membranes are in a conformation that cannot be recognized by antagonists and that membrane constraints are such that ions or guanine nucleotides cannot sufficiently modulate the conformation to allow it to recognize antagonists. On removal of membrane structure by solubilization, these constraints are removed.

Adenosine↗
PubMed1990

In vivo pertussis toxin administration: effects on the function and levels of Gi alpha proteins and their messenger ribonucleic acids.

Pertussis toxin ADP-ribosylates and functionally inactivates a number of guanine nucleotide-binding proteins (G-proteins), including the inhibitory G-protein, Gi. Once ADP-ribosylated, the subsequent duration of inactivation and fate of these proteins in cells are unknown. In the present study, therefore, we have attempted to answer some of these questions in an in vivo rat adipocyte model. Our results indicate a rather prolonged (greater than 2 weeks) inactivation of Gi, as evidenced by loss of R-phenylisopropyladenosine-mediated inhibition of adenylate cyclase activity in rat adipocyte membranes after administration of the toxin in vivo. This was associated with a complete loss of pertussis toxin ADP-ribosylatable substrates on days 3 and 15, with only partial recovery by day 28 after initiation of treatment. Specific antibodies used to quantitate the levels of alpha i1- and alpha 12-subunits of alpha i indicate a 2-fold increase in the levels of these proteins by day 3, followed by a progressive decrease and subsequent recovery in these subunits to 53%, 47%, and 87% of the control value on days 15, 20, and 28, respectively. No change in the levels of alpha s was observed at all time points tested. The levels of mRNA encoding the alpha i proteins were measured using specific oligonucleotide probes to determine whether changes in the levels of these transcripts could explain the observed regulation of alpha i. At the early time point (day 3), there was no alteration in the mRNA for alpha 3, alpha i1, alpha i2, and alpha i3 in fat pads from control and treated rats. However, the levels of the alpha i1 transcript decreased slightly in the treated groups on day 15-20 by a maximum of 33% and partially recovered by day 28. No changes in the alpha i2, alpha i3, alpha s, or tubulin transcripts were observed over the same treatment period. These results provide the first evidence that ADP ribosylation of the alpha i proteins by pertussis toxin leads to significant alterations in their levels. Furthermore, alterations in the levels of alpha i proteins (except alpha i1) do not appear to result from transcriptional regulation.

Adenosine Diphosphate Ribose↗
PubMed1990

Adenosine receptors and beyond: molecular mechanisms of physiological regulation.

Through this presentation, I have attempted to highlight some of the work that has been carried out in my laboratory over the past four years. We have strived to take a systemic approach to the study of the structure, function, and regulation of adenosine receptors and the transmembrane signalling processes that they activate. Much remains to be learned.

Animals↗
PubMed1990

Demonstration of both A1 and A2 adenosine receptors in DDT1 MF-2 smooth muscle cells.

Adenosine receptors of the A1 and A2 subtypes were characterized in membranes from DDT1 MF-2 smooth muscle cells. These cells possess a high density of A1 adenosine receptors (Bmax = 0.8-0.9 pmol/mg of protein), as measured by both agonist and antagonist radioligands. Agonists compete for [125I]N6-[2-(4-amino-3-iodophenyl)ethyl]-adenosine (A1 receptor-selective radioligand) binding with the following potency series: (R)-phenylisopropyladenosine [(R)-PIA] greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (S)-PIA, indicative of their interaction with A1 adenosine receptors. Agonist competition for [3H]8-(4-[[[(2-aminoethyl)amino]carbonyl)methyl)oxy]phenyl)-1, 3-dipropylxanthine [( 3H]XAC) (an antagonist radioligand for the A1 adenosine receptor) was described by a two-state model of 1.3 nM (high affinity state, KK) and 370 nM (low affinity state, KL), with 70% of the receptors in the high affinity state (RH). Addition of guanosine 5'-[beta, alpha-imido]triphosphate (100 microM) shifted the (R)-PIA competition curves to the right to lower affinities. Photoaffinity labeling with the agonist photoprobe [125I]N6-[2-(4-amino-3-iodophenyl) ethyl]adenosine indicates that the A1 adenosine receptor binding subunit is a Mr 38,000 protein. Adenosine receptor agonists [(R)-PIA, NECA, and (S)-PIA] inhibited isoproterenol-stimulated adenylate cyclase activity in DDT1 MF-2 cell membranes with IC50 values of 62, 538, and 750 nM, respectively. Inhibition of adenylate cyclase by (R)-PIA was attenuated by the A1 receptor antagonist XAC and following inactivation of Gi with pertussis toxin (100 ng/ml). Using a recently developed A2 adenosine receptor agonist radioligand 2-[4-(2-[( 4-aminophenyl]methylcarbonyl)ethyl) phenyl]ethylamino-5'-N-ethylcarboxamido adenosine (125I-PAPA-APEC), we have demonstrated the presence of A2 adenosine receptors in this cell line. Saturation curves with 125I-PAPA-APEC indicated the Bmax and Kd values to be 0.21 pmol/mg of protein and 4.0 nM, respectively. In competition experiments, NECA was more potent at inhibiting 125I-PAPA-APEC binding than (R)-PIA, with their respective IC50 values being 5.6 and 351 nM. The photolabeled A2 adenosine receptor migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 42,000. Finally, adenosine receptor agonists stimulated adenylate cyclase activity by approximately 2-3 fold with the following potency series: PAPA-APEC greater than or equal to NECA greater than (R)-PIA, indicative of their interaction at A2 receptors. These data represent the first demonstration of the presence of both A1 and A2 receptors in a single cell line, DDT1 MF-2 smooth muscle cells.

Adenylyl Cyclases↗
PubMed1990

Glycoprotein nature of the A2-adenosine receptor binding subunit.

Mammalian A2-adenosine receptor binding subunits (A2AR) can be visualized by covalent labeling with the photoaffinity crosslinking ligand 125I-2-[4-[2-[2-[(4-aminophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl]ethylamino-5'-N-ethylcarboxamidoad enosine or directly with the azide derivative described in this paper. The protein comprising the A2-adenosine receptor binding subunit migrates with a Mr of 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the glycoproteins representing the radiolabeled A1- and A2-adenosine receptor binding subunit from bovine brain were compared by partial peptide maps and following treatment with exo- and endoglycosidases. Peptide maps using two separate proteases reveal that the A1- and A2-adenosine receptor binding subunits share no common peptide fragments by two-dimensional gel electrophoresis. Endoglycosidase F treatment of labeled A2AR results in a single labeled peptide of Mr 38,000 without intermediate peptides, suggesting a single N-linked carbohydrate chain. The labeled A2AR demonstrates a sensitivity to neuraminidase, as evidenced by an increased mobility on gel electrophoresis, suggesting the receptors contain a glycan component containing terminal sialic acid. Treatment of the labeled A2AR with alpha-mannosidase reveals two distinct populations of A2ARs, one of which is sensitive and the other resistant to the enzyme. The nonadditivity of sequential treatments with the two exoglycosidases suggests, a heterogeneous population of A2AR containing either complex- or high mannose-type carbohydrate chains. These data suggest the A2AR is a Mr 45,000 glycoprotein with a single carbohydrate chain of either the complex or high mannose type. In addition, the A1- and A2ARs are distinct glycoproteins, as evidenced by their differing molecular weights (before and after deglycosylation) and distinct peptide maps.

Adenosine↗
PubMed1989

Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor.

An approximate 140-fold purification of the A1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi-Gel 10 coupled through an amide linkage to XAC, a high-affinity A1 adenosine receptor antagonist. As assessed by [3H]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 pmol/mg protein. Interaction of solubilized A1 adenosine receptors with the XAC-Affi-Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [3H]XAC binding in the material eluted from the column with R-PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 +/- 22 pmol/mg protein with typically 5-15% of the bound receptor recovered. The purified receptor displayed high-affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A1 adenosine receptor: R-PIA greater than S-PIA greater than NECA. In purified preparations, the photoaffinity probe [125I]PAPAXAC-SANPAH specifically labelled a protein of molecular mass 38,000 which has previously been shown to be the A1 adenosine receptor binding subunit.

Animals↗
PubMed1989

Demonstration of distinct agonist and antagonist conformations of the A1 adenosine receptor.

A1 adenosine receptor-binding subunits can be visualized using high affinity antagonist and agonist photoaffinity radioligands. In the present study, we examined whether agonists and antagonists bind to the same receptor-binding subunit and if agonists and antagonists induce different conformational states of the receptor in intact membranes. It was demonstrated that several agonist and antagonist photoaffinity receptor-binding subunit. When the agonist and antagonist photoaffinity labeled peptides were denatured and subjected to partial peptide map analysis using a two-dimensional gel electrophoresis system similar peptide fragments were generated from each specifically labeled protein. This suggests that both classes of ligand label and incorporate into the same binding subunit. Proteolytic digestions of agonist- and antagonist-occupied receptors in native intact membranes revealed distinct and different peptide fragments depending on whether the ligand was an agonist or an antagonist. Manipulation of incubation conditions to perturb ligand-receptor interactions alter the pattern of peptide fragments generated with each specific protease. These data suggest that agonist and antagonist photoaffinity probes interact with an incorporate into the same binding subunit but that agonist binding is associated with a unique and detectable receptor conformation.

Affinity Labels↗
PubMed1989

Agonist derived molecular probes for A2 adenosine receptors.

The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5'-N- ethylcarboxamidoadenos ine (CGS21680) was recently reported to be selective for the A2 adenosine receptor subtype, which mediates its hypotensive action. To investigate structure/activity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A2 adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A2 receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2 receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology.

Adenosine↗
PubMed1989

Altered thyroid status regulates the adipocyte A1 adenosine receptor-adenylate cyclase system.

To assess the effect of hyperthyroidism on the adenosine receptor-adenylate cyclase system in adipocytes, membranes from hyperthyroid and control rats were prepared. Rats were rendered hyperthyroid by five days of injection with triiodothyronine (T3). Basal as well as isoproterenol-, sodium fluoride-, forskolin- and manganese (Mn++)-stimulated adenylate cyclase activities are attenuated 20-30% in adipocyte membranes from hyperthyroid animals. There is a greater inhibition of total adenylate cyclase activity in response to R-PIA, A1 selective inhibitory agonist, in membranes from hyperthyroid animals. However, on a percentage basis, R-PIA is equally effective at inhibiting adenylate cyclase activity in control and treated membranes. Using antagonist radioligands, [3H]XAC (A1 receptor) and [125I]CYP (beta-adrenergic receptor), no significant alteration in receptor number is observed in hyperthyroidism. In addition, no alteration in Gi protein-A1 receptor coupling is noted as exhibited by R-PIA competition curves. These findings suggest hyperthyroidism most likely results in a decrease of the catalytic moiety of adenylate cyclase either quantitatively or functionally.

Adenylyl Cyclases↗
PubMed1989

Electrophilic derivatives of purines as irreversible inhibitors of A1 adenosine receptors.

Functionalized congeners derived from 1,3-dipropyl-8-phenylxanthine and from N6-phenyladenosine were derivatized to contain electrophilic groups (isothiocyanate, N-hydroxysuccinimide ester, maleimide, sulfonyl chloride, or alpha-haloacyl group) capable of reaction with nucleophiles on biopolymers. The goal was to inhibit chemically the A1 adenosine receptor by using reactive agonist and antagonist ligands. Some of the electrophilic derivatives were synthesized through acylation of amine-functionalized congeners using hetero- or homobifunctional reagents available for protein cross-linking. The affinity for A1 adenosine receptors was evaluated in competitive binding assays by using rat and bovine brain membranes. Several xanthine and adenosine thiourea derivatives prepared from 1,3- and 1,4-phenylene diisothiocyanate (DITC) were potent irreversible inhibitors of adenosine receptors. Derivatives of m-DITC, at concentrations between 10 and 500 nM, irreversibly eliminated binding at 90% of the A1-receptor sites. Receptor affinity of both xanthine and adenosine derivatives containing distal phenylthiourea substituents was diminished by electron-donating groups on the ring.

Adenosine↗
PubMed1989

Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking.

A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors (see above) and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate. Photoaffinity crosslinking of the A1 adenosine receptor binding subunit with 125I-labeled 8-[4-[2-(4- aminophenylacetylamino)ethyl]carbonylmethyloxyphenyl]-1,3-di propylxanthine (PAPAXAC) (an A1 selective photoaffinity probe) in the same tissue reveals a 38-kDa peptide that exhibits the appropriate A1 receptor pharmacology. 125I-labeled PAPA-APEC, therefore, has identified the A2 receptor binding subunit as a 45-kDa protein that is unique and distinct from the A1 binding subunit.

Adenosine↗
PubMed1989

Mechanisms of receptor activation in adenylate cyclase systems.

Recent years have seen a dramatic increase in our knowledge of the structure, function, and regulation of hormone-sensitive adenylate cyclase systems. The detailed structure of receptors and guanine nucleotide binding proteins (G proteins) are now known. Biochemical techniques for the in vitro reconstitution of purified components have facilitated the study of how these components interact to promote the transmembrane signalling process. It is now clear that agonists but not antagonists induce a whole series of interactions beginning with protein conformational changes in the receptor followed by activation of a specific guanine nucleotide binding protein consequent to GTP binding. The activated G protein ultimately results in activation of the enzyme adenylate cyclase with the catalysis of ATP to cAMP. A multitude of studies have demonstrated that each of these components and their interactions can be modulated by a variety of pathophysiological states. Partial agonists represent a class of drugs that can fully occupy receptors, yet induce a less than maximal response compared to full agonists. The mechanism by which these agents produce a less than full response remains unclear, but several possibilities exist. These putative mechanisms, as well as the role of partial agonists in regulating adenylate cyclase systems, will be discussed in detail.

Adenylyl Cyclases↗
PubMed1989

Modification of the rat adipocyte A1 adenosine receptor-adenylate cyclase system during chronic exposure to an A1 adenosine receptor agonist: alterations in the quantity of GS alpha and Gi alpha are not associated with changes in their mRNAs.

The A1-adenosine receptor (A1AR) adenylate cyclase system in rat adipocytes undergoes heterologous desensitization following chronic in vivo exposure to an A1AR agonist (+)-N6-(R-phenylisopropyl)adenosine [J. Biol. Chem. 262:841-847 (1987)]. This desensitization involves an absolute increase in adenylate cyclase activity and a refractoriness to receptor ligands that are inhibitory to adenylate cyclase. In this study, receptor changes were characterized using an A1AR antagonist radioligand, [3H]8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropyl xanthine. Saturation binding studies demonstrated a 47% decrease in total A1AR density without a change in KD. Agonist competition studies revealed a decreased percentage of receptors, from 55% to 35%, in the high affinity state following desensitization. An increase in GS alpha of 49% was found by Western blotting using specific GS alpha antibodies. Further, an antibody that recognizes Gi alpha 1 adn Gi alpha 2 was used to quantitate these subtypes of Gi alpha and both were decreased by 59% following desensitization. However, when an antibody that recognizes Gi alpha 3 was used, no change in Gi alpha 3 was found, demonstrating, in this case, differential regulation of Gi alpha subtypes. The mechanisms responsible for changes in GS alpha and Gi alpha were studied by measuring the levels of their mRNAs from normal and desensitized adipocytes. Using either labeled cDNAs (Gi alpha 2, Gi alpha 3) or oligonucleotides (GS alpha, Gi alpha 1), Northern analysis demonstrated that mRNAs for GS alpha and all three isoforms of Gi alpha are present in adipocytes but that there are no changes in the levels of any of these transcripts following desensitization. These data suggest that desensitization of the A1AR-adenylate cyclase system involves a down-regulation of A1ARs and an additional loss of A1AR agonist high affinity sites. Further, an increase in GS alpha, a decrease in Gi alpha 1 and Gi alpha 2, and no change in Gi alpha 3 were found. The regulation of GS alpha and the subtypes of Gi alpha in this system does not occur by altering the levels of their respective transcripts.

Adenylyl Cyclases↗
PubMed1988

A novel site of action of a high affinity A1 adenosine receptor antagonist.

XAC, a high affinity antagonist of the A1 adenosine receptor, enhances adenylate cyclase activity by 1.3-2 fold with an EC50 of approximately 47 nM in adipocyte membranes pretreated with adenosine deaminase to eliminate adenosine and in the presence of total phosphodiesterase inhibition by 100 microM papaverine. This effect of XAC is observed only at concentrations of GTP sufficient to activate Gi (approximately 5 x 10(-6) M GTP) and is not evident in the absence or presence of lower GTP concentrations. ADP ribosylation of Gi by pertussis toxin treatment also abolishes this stimulatory action of XAC. Furthermore, in the presence of GTP activation of inhibitory prostaglandin E1 receptors diminishes the stimulatory effect of XAC on adenylate cyclase. In addition, XAC interferes with GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity in a noncompetitive manner. Finally, XAC is only a weak inhibitor of the low Km cyclic AMP phosphodiesterase, producing approximately 40% inhibition of phosphodiesterase activity at a concentration of 100 microM. These data suggest that XAC increases adenylate cyclase activity in absence of endogenous adenosine by inhibiting tonic Gi activity in a reversible manner.

Adenylyl Cyclases↗
PubMed1988

A1 adenosine receptor-G protein coupling in bovine brain membranes: effects of guanine nucleotides, salt, and solubilization.

The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAC) but not agonist (R-N6-[3H]phenylisopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Heart rate control in diabetic rabbits: blunted response to isoproterenol.

To study the effects of chronic diabetes on heart rate and adrenergic responsiveness we compared unanesthetized diabetic rabbits, 10-13 mo after alloxan monohydrate injection, to age-matched controls. There were no significant differences found between groups for body or heart weight. Both resting and intrinsic heart rate (the latter obtained after atropine sulfate and propranolol HCl) were similar. In addition, serum and left ventricular epinephrine and norepinephrine concentrations as well as left ventricular beta-receptor density and affinity were unchanged in diabetic animals. Heart rate responses to isoproterenol were blunted in diabetics at the three highest doses. Base-line mean blood pressure was modestly lower in diabetic rabbits, and parallel declines in pressure for both groups were observed in response to isoproterenol. The diminished heart rate response to isoproterenol in diabetic rabbits may be due to diminished myocardial sensitivity to catecholamines, possibly combined with altered baroreceptor reflexes. These experiments may provide an explanation for the blunted heart rate response to exercise described in human diabetics.

Animals↗